Preoperative evaluation of mammographic microcalcifications after neoadjuvant chemotherapy for breast cancer.

AIM: To assess the predictive value of preoperative residual mammographic microcalcifications for residual tumours after neoadjuvant chemotherapy (NAC) for breast cancer. MATERIALS AND METHODS: This single-centre retrospective study included breast cancer patients who underwent NAC and demonstrated suspicious microcalcifications within or near the tumour bed on mammography from June 2015 to August 2018. The residual microcalcifications and remnant lesion on magnetic resonance imaging (MRI) were correlated with histopathological findings of residual tumours and immunohistochemical markers. RESULTS: A total of 96 patients were included. Ten patients achieved pathological complete response (pCR) and previous suspicious microcalcifications were associated with benign pathology in 10.4% (10/96) of the patients. In the remaining 86 patients who did not achieve pCR, 61.5% (59/96) of the residual microcalcifications were associated with invasive or in situ carcinoma and 28.1% (27/96) with benign pathology. Hormone receptor-positive (HR+) patients had the highest proportion of residual malignant microcalcifications compared to HR- patients (48.9% versus 13.5%, respectively; p=0.019). MRI correlated better than residual microcalcifications on mammography in predicting residual tumour extent in all subtypes (ICC=0.709 versus 0.365). MRI also showed higher correlation with residual tumour size for the HR-/HER2+ and HR-/HER2- subtype (ICC=0.925 and 0.876, respectively). CONCLUSION: The extent of microcalcifications on mammography after NAC did not correlate with the extent of residual cancer in 38.5% of women. Regardless of the extent of microcalcifications, residual tumour extent on MRI after NAC and molecular subtype could be an accurate tool in evaluating residual cancer after NAC.

Hepatic inducible nitric oxide synthase expression increases upon exposure to hypergravity

Stimulation by a number of conditions, including infection, cytokines, mechanical injury, and hypoxia, can upregulate inducible nitric oxide synthase (iNOS) in hepatocytes. We observed that exposure to hypergravity significantly upregulated the transcription of the hepatic iNOS gene. The aim of this study was to confirm our preliminary data, and to further investigate the distribution of the iNOS protein in the livers of mice exposed to hypergravity. ICR mice were exposed to +3 Gz for 1 h. We investigated the time course of change in the iNOS expression. Hepatic iNOS mRNA expression progressively increased in centrifuged mice from 0 to 12 h, and then decreased rapidly by 18 h. iNOS mRNA levels in the livers of centrifuged mice was significantly higher at 3, 6, and 12 h than in uncentrifuged control mice. The pattern of iNOS protein expression paralleled that of the mRNA expression. At 0 and 1 h, weak cytoplasmic iNOS immunoreactivity was found in some hepatocytes surrounding terminal hepatic venules. It was noted that at 6 h there was an increase in the number of perivenular hepatocytes with moderate to strong cytoplasmic immunoreactivity. The number of iNOS-positive hepatocytes was maximally increased at 12 h. The majority of positively stained cells showed a strong intensity of iNOS expression. The expression levels of iNOS mRNA and protein were significantly increased in the livers of mice exposed to hypergravity. These results suggest that exposure to hypergravity significantly upregulates iNOS at both transcriptional and translational levels.

SOX2 regulates self-renewal and tumorigenicity of stem-like cells of head and neck squamous cell carcinoma.

BACKGROUND: Head and neck squamous cell carcinomas (HNSCCs) display cellular heterogeneity and contain cancer stem cells (CSCs). Sex-determining region Y [SRY]-box (SOX)2 is an important regulator of embryonic stem cell fate and is aberrantly expressed in several types of human tumours. Nonetheless, the role of SOX2 in HNSCC remains unclear. METHODS: We created cells ectopically expressing SOX2 from previously established HNSCC cells and examined the cell proliferation, self-renewal capacity, and chemoresistance of these cells compared with control cells. In addition, we knocked down SOX2 in primary spheres obtained from HNSCC tumour tissue and assessed the attenuation of stemness-associated traits in these cells in vitro and in vivo. Furthermore, we examined the clinical relevance of SOX2 expression in HNSCC patients. RESULTS: SOX2 is aberrantly expressed in primary tissue of HNSCC patients but not in healthy tissue. SOX2 expression correlated with tumour recurrence and poor prognosis of HNSCC patients. Ectopic expression of SOX2 induced cell proliferation via cyclin B1 expression and stemness-associated features, such as self-renewal and chemoresistance. In addition, a knockdown of SOX2 in HNSCC CSCs attenuated their self-renewal capacity, chemoresistance (through ABCG2 suppression), invasion capacity (via snail downregulation), and in vivo tumorigenicity. CONCLUSIONS: These results suggest that SOX2 may have important roles in the 'stemness' and progression of HNSCC. Targeting SOX2-positive tumour cells (CSCs) could be a new therapeutic strategy in HNSCCs.

Hepatic inducible nitric oxide synthase expression increases upon exposure to hypergravity.

Stimulation by a number of conditions, including infection, cytokines, mechanical injury, and hypoxia, can upregulate inducible nitric oxide synthase (iNOS) in hepatocytes. We observed that exposure to hypergravity significantly upregulated the transcription of the hepatic iNOS gene. The aim of this study was to confirm our preliminary data, and to further investigate the distribution of the iNOS protein in the livers of mice exposed to hypergravity. ICR mice were exposed to +3 Gz for 1 h. We investigated the time course of change in the iNOS expression. Hepatic iNOS mRNA expression progressively increased in centrifuged mice from 0 to 12 h, and then decreased rapidly by 18 h. iNOS mRNA levels in the livers of centrifuged mice was significantly higher at 3, 6, and 12 h than in uncentrifuged control mice. The pattern of iNOS protein expression paralleled that of the mRNA expression. At 0 and 1 h, weak cytoplasmic iNOS immunoreactivity was found in some hepatocytes surrounding terminal hepatic venules. It was noted that at 6 h there was an increase in the number of perivenular hepatocytes with moderate to strong cytoplasmic immunoreactivity. The number of iNOS-positive hepatocytes was maximally increased at 12 h. The majority of positively stained cells showed a strong intensity of iNOS expression. The expression levels of iNOS mRNA and protein were significantly increased in the livers of mice exposed to hypergravity. These results suggest that exposure to hypergravity significantly upregulates iNOS at both transcriptional and translational levels.

Differential and cell-type specific microheterogeneity of high mannose-type Asn-linked oligosaccharides of human transferrin receptor.

The structural analysis of high mannose-type Asn-linked (N-linked) oligosaccharides of the human transferrin receptor (hTR) from D-[2-3H]mannose metabolic-radiolabeled human cells--A431, K562, BeWo, and HL60--was investigated. The radiolabeled hTR glycopeptides were prepared and fractionated by a lectin chromatography of Concanavalin A-Sepharose. The composition analysis of hTR glycopeptides revealed that Con A-I contains both mannose and fucose, whereas Con A-III has mannose exclusively. The Con A-III glycopeptides were treated with Endo H. The released oligosaccharides were charge-fractionated by QAE-Sephadex. The neutral oligosaccharides were further size-fractionated by an amine absorption high performance liquid chromatography (HPLC). Our results demonstrate that the high mannose-type oligosaccharides of hTR ranged in size from Man5-R to Man9-R with cell-type specific patterns. A relative amount of each component was found to be differentially heterogeneous among the four different human cell lines.

The hepatitis B virus-X protein activates a phosphatidylinositol 3-kinase-dependent survival signaling cascade.

The hepatitis B virus-X (HBx) protein is known as a multifunctional protein that not only coactivates transcription of viral and cellular genes but coordinates the balance between proliferation and programmed cell death, by inducing or blocking apoptosis. In this study the role of the HBx protein in activation of phosphatidylinositol 3-kinase (PI3K) was investigated as a possible cause of anti-apoptosis in liver cells. HBx relieved serum deprivation-induced and pro-apoptic stimuli-induced apoptosis in Chang liver (CHL) cells. Treatment with 1-d-3-deoxy-3-fluoro-myo-inositol, an antagonist to PI3K, which blocks the formation of 3'-phosphorylated phosphatidyl inositol in CHL cells transformed by HBx (CHL-X) but not normal Chang liver (CHL) cells, showed a marked loss of viability with evidence of apoptosis. Similarly, treatment with wortmannin, an inhibitor of PI3K, stimulated apoptosis in HBx-transformed CHL cells but not in normal cells, confirming that HBx blocks apoptosis through the PI3K pathway. The serine 47 threonine kinase, Akt, one of the downstream effectors of PI3K-dependent survival signaling was 2-fold higher in HBx-transformed CHL (CHL-X) cells than CHL cells. Phosphorylation of Akt at serine 473 and Bad at serine 136 were induced by HBx, which were specifically blocked by wortmannin and dominant negative mutants of Akt and Bad, respectively. We also demonstrated that HBx inhibits caspase 3 activity and HBx down-regulation of caspase 3 activity was blocked by the PI3K inhibitor. Regions required for PI3K phosphorylation on the HBx protein overlap with the known transactivation domains. HBx blocks apoptosis induced by serum withdrawal in CHL cells in a p53-independent manner. The results indicate that, unlike other DNA tumor viruses that block apoptosis by inactivating p53, the hepatitis B virus achieves protection from apoptotic death through a HBx-PI3K-Akt-Bad pathway and by inactivating caspase 3 activity that is at least partially p53-independent in liver cells. Moreover, these data suggest that modulation of the PI3K activity may represent a potential therapeutic strategy to counteract the occurrence of apoptosis in human hepatocellular carcinoma.

Novel induction of alpha-lactalbumin-mediated lacdiNAc-R expression in vivo.

alpha-Lactalbumin (alpha-LA) is a regulatory protein by which the mammalian beta1,4-galactosyltransferase (beta1,4-galT) is induced to utilize glucose as an acceptor instead of N-acetylglucosamine (GlcNAc) during lactose synthesis in mammary gland. alpha-LA can also modulate beta1,4-galT to utilize UDP-N-acetylgalactosamine (UDP-GalNAc) as a donor towards GlcNAc acceptor substrate with high efficiency in vitro [Do, Do and Cummings (1995) J. Biol. Chem. 270, 18447-18451]. In the present study we transfected cDNA encoding bovine alpha-LA into Lec8 cells and examined whether nucleotide sugar switching of UDP-galactose (UDP-Gal) into UDP-GalNAc occurred in vivo and whether the neo-glycosylation of GalNAcbeta1,4GlcNAc-R structure was synthesized in alpha-LA-stable transfectants. Our studies demonstrate that the stable expression of alpha-LA in Lec8 cells induces the formation of GalNAcbeta1,4GlcNAc-R in vivo through the nucleotide sugar switching of beta1,4-galT.

Akt protein kinase enhances human telomerase activity through phosphorylation of telomerase reverse transcriptase subunit.

With the amino acid sequences of all reported Akt kinase physiological substrates, the possible Akt kinase substrate specificity has been suggested. The serine/threonine residue to be phosphorylated in these proteins is placed within stretches of amino acids with homology, and the arginine residues on the -5 and -3 positions and a hydrophobic amino acid on the +2 position are conserved relative to those of serine/threonine residues (XXRXRXXS/TXX). We noticed two putative Akt kinase phosphorylation sites (220GARRRGGSAS229) and (817AVRIRGKSYV826) in human telomerase reverse transcriptase (hTERT) subunit. To demonstrate that hTERT is an Akt kinase substrate protein, we performed the nonradioactive protein kinase assay with the fluorescein hTERT peptide (817AVRIRGKSYV826). We observed the phosphorylation of hTERT peptide by the human melanoma cell lysate or the activated recombinant Akt kinase proteins in vitro. With the treatment of the growth factor deprivation or okadaic acid, we also observed the up-regulation of both hTERT peptide phosphorylation and the telomerase activity. We noticed that Wortmannin down-regulates hTERT peptide phosphorylation and telomerase activity together. In addition, we observed the enhancement of telomerase activity with the pretreatment of Akt kinase in vitro. Thus, these observations suggest that Akt kinase enhances human telomerase activity through phosphorylation of hTERT subunit as one of its substrate proteins.

Cloning and expression of cDNA for a human Sia alpha 2,3Gal beta 1, 4GlcNA:alpha 2,8-sialyltransferase (hST8Sia III).

The cDNA encoding human Sia-alpha2,3-Gal-beta1,4-GlcNAc-R:alpha2, 8-sialyltransferase, hST8Sia III, was isolated by screening of a human brain cDNA library with polymerase chain reaction-amplified DNA probe generated from the sequence of mouse ST8Sia III (mST8Sia III) and by 5' rapid amplification of cDNA ends of mRNA isolated from human brain tissues. Comparative analysis of the predicted protein-coding region between our cloned hST8Sia III and mST8Sia III showed 92 and 96% identities in the nucleotide and the amino acid sequence, respectively. The soluble hST8Sia III protein expressed in COS-7 showed an extremely high catalytic activity of transferring sialic acid through alpha2,8-linkage to intact fetuin glycoprotein, whereas the transferring activity was completely undetectable toward either alpha2,6-sialylated glycoprotein or desialylated glycoprotein acceptors. Northern analysis of hST8Sia III showed that the transcript corresponding to 11 kb was expressed in both human fetal and adult brain, while the expression of the 5.5-kb transcript was restricted to fetal liver, indicating that the expression of hST8Sia III is developmentally and tissue-specifically regulated.

Jacalin interacts with Asn-linked glycopeptides containing multi-antennary oligosaccharide structure with terminal alpha-linked galactose.

The carbohydrate binding properties of jacalin lectin were examined using RAF9 cell-derived D-[6-3H]glucosamine-radiolabeled total glycopeptides containing N-linked and O-linked oligosaccharides. The binding of N-linked glycopeptides to jacalin was abolished by treatment of alpha-galactosidase whereas O-linked glycopeptides were still bound lectin after this treatment. The removal of O-linked oligosaccharides by mild alkaline/borohydride treatment completely eliminated the lectin binding of alpha-galactosidase treated glycopeptides. These results demonstrate that jacalin interacts with cellular glycopeptides containing N-linked oligosaccharides with terminal alpha-galactose residues as well as glycopeptides containing O-linked oligosaccharides.

Differential expression of LacdiNAc sequences (GalNAc beta 1-4GlcNAc-R) in glycoproteins synthesized by Chinese hamster ovary and human 293 cells.

The lacdiNAc sequence GalNAc beta 1-->4GlcNAc beta 1-R occurs in the N- and O-glycans of many glycoproteins in vertebrate and invertebrates. We now report that both human 293 cells and Chinese hamster ovary (CHO) cells contain a UDPGalNAc:GlcNAc beta 1,4 N-acetylgalactosaminyltransferase (beta 1,4GalNAcT) that forms the lacdiNAc sequence. The beta 1,4GalNAcT in CHO cells is distinct from beta 1,4 galactosyltransferase in that the latter enzyme, but not the former, binds to a column of immobilized bovine alpha-lactalbumin. To determine whether endogenous glycoproteins in these cells contain lacdiNAc sequences, glycoproteins from 293 cells, CHO and Lec8 CHO cells were desialylated and passed over immobilized Wisteria floribunda agglutinin (WFA), a plant lectin with affinity for terminal GalNAc residues. WFA bound to approximately 120 and approximately 80 kDa glycoproteins in 293 cells and glycans from these glycoproteins contained lacdiNAc sequences. The approximately 120 kDa glycoproteins in 293 cells bound by WFA is a mixture of both the lysosome-associated membrane glycoproteins LAMPs-1 and -2. WFA bound to two glycoproteins of approximately 47 and approximately 78 kDa in Lec8 CHO cells, but these glycoproteins are not LAMPs and they do not contain the lacdiNAc sequence. Instead, they contain multiple GalNAc alpha-Ser/Thr O-glycans that promote binding to WFA. Thus, the beta 1,4GalNAcT in 293 cells displays a limited specificity in its recognition of acceptors, whereas the beta 1,4GalNAcT in CHO cells fails to promote synthesis of the cognate lacdiNAc sequence. The presence of the beta 1,4GalNAcT may not be sufficient for synthesis of lacdiNAc sequences and other factors may contribute to regulate the functionality of the enzyme.

Immobilized Lotus tetragonolobus agglutinin binds oligosaccharides containing the Le(x) determinant.

A defined set of oligosaccharides and glycopeptides containing alpha-linked fucose were used to examine the specificity of the immobilized fucose-binding lectin Lotus tetragonolobus agglutinin (LTA1), also known as lotus lectin. Glycans containing the Lewis x determinant (Le(x)) Gal beta 1-4[Fuc alpha 1-3]GlcNAc beta 1-3-R were significantly retarded in elution from high density LTA-Emphaze columns. The lectin also bound the fucosylated lacdiNAc trisaccharide GalNAc beta 1-4[Fuc alpha 1-3]GlcNAc. The lectin did not bind glycans containing either sialylLe(x) or VIM-2 determinants, nor did it bind the isomeric Le(x), Gal beta 1-3[Fuc alpha 1-4]GlcNAc-R. Although 2'-fucosyllactose Fuc alpha 1-2Gal beta 1-4Glc) was retarded in elution from the columns, larger glycans containing the H-antigen Fuc alpha 1-2Gal beta 1-3(4)GlcNAc-R interacted poorly with immobilized LTA. Our results demonstrate that immobilized LTA is effective in isolating glycans containing the Le(x) antigen and is useful in analyzing specific fucosylation of glycoconjugates.

Alpha-lactalbumin induces bovine milk beta 1,4-galactosyltransferase to utilize UDP-GalNAc.

We now report that alpha-lactalbumin (alpha-LA) has a novel effect on bovine milk UDP-Gal:GlcNAc-beta 1,4-galactosyltransferase (beta 1,4-GT) and induces the enzyme to efficiently utilize UDP-GalNAc as a donor. In the presence of alpha-LA the enzyme transfers GalNAc to free GlcNAc to produce GalNAc beta 1-4GlcNAc at a rate 55% of that compared to the rate when UDP-Gal is the donor in the absence of alpha-LA. The stimulation by alpha-LA is dependent on the concentrations of alpha-LA, acceptor, and sugar nucleotide. Interestingly, beta 1,4-GT is unable to transfer Gal-NAc to Glc with or without alpha-LA. alpha-LA also stimulates the transfer of GalNAc from UDP-GalNAc to various chitin oligomers, although the degree of stimulation decreases as the acceptor size increases. Thus, bovine milk beta 1,4-GT has an inherent ability to utilize two different sugar nucleotides and the sugar nucleotide preference is regulatable by alpha-LA.

Presence of O-linked oligosaccharide on a threonine residue in the human transferrin receptor.

We have previously demonstrated that the human transferrin receptor (TfR) of approximately 90 kDa contains Ser/Thr-linked (O-linked) oligosaccharides. In the present study, we report our identification of the site of attachment of the O-linked oligosaccharides in the receptor. A 70 kDa fragment from the external domain of the TfR was generated by trypsin treatment of the [3H]glucosamine-labelled receptor purified from human K562 cells. The beta-elimination of the intact TfR, but not the 70 kDa fragment, released Gal-[3H]Gal-NAcitol, indicating that the 70 kDa fragment lacks O-linked oligosaccharides. In the remaining 20 kDa fragment there are three potential sites (Thr96, Thr104 and Ser106) for O-glycosylation in the extracellular domain. To identify which of these residues are O-glycosylated, both the [3H]Thr- and [3H]Ser-labelled TfR were directly treated with mild base to effect beta-elimination, and the radiolabelled amino acids and their derivatives were analysed. Approximately 2% of the total radiolabelled Thr, but no radiolabelled Ser, was converted to expected beta-elimination products by this treatment. These and other results demonstrate that only one O-linked oligosaccharide is present in the TfR and that it occurs on either Thr96 or Thr104. From human serum we purified the cleaved, soluble form of the TfR (s-TfR), which contains Thr104, but lacks Thr96. The s-TfR was sensitive to O-glycanase and bound to Jacalin lectin, indicating that the s-TfR contains an O-linked oligosaccharide.(ABSTRACT TRUNCATED AT 250 WORDS)

The hamster transferrin receptor contains Ser/Thr-linked oligosaccharides: use of a lectin-resistant CHO cell line to identify glycoproteins containing these linkages.

We recently reported that the human transferrin receptor (TfR) contains O-linked GalNAc residues [1]. To investigate whether this modification is shared by transferrin receptors in other mammals, we investigated the glycosylation of TfR in hamster cells. To facilitate our analysis the lectin-resistant Chinese hamster ovary (CHO) cell line Lec8 was used. These cells are unable to galactosylate glycoproteins, resulting in truncation of the Ser/Thr-linked oligosaccharides to a single residue of terminal alpha-linked GalNAc. This structure is bound with high affinity by the lectin Helix pomatia agglutinin (HPA). The TfR was affinity purified from Lec8 cells metabolically radiolabeled with [3H]glucosamine and the receptor was found to bind tightly to HPA-Sepharose. Treatment of the purified TfR with mild alkaline/borohydride released [3H]GalNAcitol, demonstrating the presence of O-linked GalNAc. We also found that many other unidentified [3H]glucosamine-labeled glycoproteins from Lec8 cells were bound by HPA-Sepharose. The bound and unbound glycoproteins were separated by SDS/PAGE and individual species were selected for treatment with mild base/borohydride. Treatment of glycoproteins bound by HPA, but not those unbound, resulted in the release of [3H]GalNAcitol. These studies demonstrate both that the hamster TfR contains O-linked oligosaccharides and that this approach may have general utility for identifying the presence of these oligosaccharides in other glycoproteins.

Acquisition of the functional properties of the transferrin receptor during its biosynthesis.

The properties of the newly synthesized and partially glycosylated forms of the transferrin receptor were examined to determine which co- and post-translational modifications are necessary for the acquisition of transferrin binding activity and transport of the receptor to the cell surface. The nascent transferrin receptor containing core-glycosylated asparagine-linked oligosaccharides does not possess complete intersubunit disulfide bonds, sediments predominantly as a monomer in sucrose density gradients, and shows reduced binding to transferrin-agarose. Within 20-30 min after synthesis, the transferrin receptor acquires the ability to bind to a transferrin-linked affinity column. Intersubunit disulfide bond formation occurs slowly throughout the transit of the receptor to the cell surface. These results indicate that core glycosylation of the receptor may be necessary but is not sufficient for the acquisition of the ability of the receptor to bind transferrin and that intersubunit disulfide bond formation is a post-translational event. Inhibition of complex carbohydrate synthesis by either swainsonine (1 micrograms/ml) or deoxynojirimycin (4 mM) does not inhibit the ability of this receptor to form intersubunit disulfide bonds or to be transported to the cell surface. The partially glycosylated receptor, however, does show an approximately 3-fold reduced affinity for transferrin.

Human transferrin receptor contains O-linked oligosaccharides.

We have investigated the oligosaccharides in the human transferrin receptor from three different cell lines. During our studies on the structures of the N-linked oligosaccharides of the receptor, we discovered that the receptor contains O-linked oligosaccharides. This report describes the isolation and characterization of these O-linked oligosaccharides. Three different human cell lines--K562, A431, and BeWo--were grown in media containing either [2-3H] mannose or [6-3H]glucosamine. The newly synthesized and radiolabeled transferrin receptors were purified by immunoprecipitation from cell extracts and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The receptor was proteolytically digested or treated directly with mild base/borohydride. The released radiolabeled glycopeptides and oligosaccharides were separated by a variety of chromatographic techniques, and their structures were analyzed. The transferrin receptor from all three cell types contains O-linked oligosaccharides that are released from peptide by mild base/borohydride treatment. The receptor from K562 cells contains at least one O-linked oligosaccharide having two sialic acid residues and a core structure of the disaccharide galactose-N-acetyl-galactosamine. In contrast, the O-linked oligosaccharides in the transferring receptors from both A431 and BeWo cell lines are not as highly sialylated and were identified as both the neutral disaccharide galactose-N-acetylgalactosamine and the neutral monosaccharide N-acetylgalactosamine. In addition, the receptors from all three cell lines contain both complex-type and high mannose-type N-linked oligosaccharides. The complex-type chains in the receptor from A431 cells have properties of blood group A antigens, whereas oligosaccharides in receptors from both BeWo and K562 cells lack these properties. These results are interesting since both A431 and BeWo cells, but not K562 cells, are positive for blood group A antigens. Thus, our results demonstrate that the human transferrin receptor contains O-linked oligosaccharides and that there are differences in the structures of both the O-linked and complex-type N-linked oligosaccharides on the receptors synthesized by different cell types.